It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare fo...It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells). Fibroblast and NSC-34 cell’s mitochondria are co-cultured with NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast’s mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria successfully enter to 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. In summary, mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are potential mitochondrial donor for mitochondrial transplantation study in human neurodegenerative diseases.展开更多
Retinoid X receptor a （RXRα） and its N-terminally trun- cated version tRXRα play important roles in tumorige. nesis, while some RXRg ligands possess potent anti- cancer activities by targeting and modulating the t...Retinoid X receptor a （RXRα） and its N-terminally trun- cated version tRXRα play important roles in tumorige. nesis, while some RXRg ligands possess potent anti- cancer activities by targeting and modulating the tumorigenic effects of RXRo and tRXRa. Here we describe NSC-640358 （N-6）, a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRa and inhibits the transactivation of RXRα homod- imer and RXRa/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRa, essential for 9-cis-retinoic acid binding and activating RXRg transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra w-w stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRa-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor a （TNFα）-induced AKT activation and stimulates TNFa-mediated apoptosis in cancer cells in an RXRa/tRXRo dependent manner.The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRa to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRa ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFa-mediated cancer cell apoptosis by targeting RXRα/tRXRα.展开更多
文摘It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells). Fibroblast and NSC-34 cell’s mitochondria are co-cultured with NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast’s mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria successfully enter to 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. In summary, mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are potential mitochondrial donor for mitochondrial transplantation study in human neurodegenerative diseases.
文摘Retinoid X receptor a （RXRα） and its N-terminally trun- cated version tRXRα play important roles in tumorige. nesis, while some RXRg ligands possess potent anti- cancer activities by targeting and modulating the tumorigenic effects of RXRo and tRXRa. Here we describe NSC-640358 （N-6）, a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRa and inhibits the transactivation of RXRα homod- imer and RXRa/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRa, essential for 9-cis-retinoic acid binding and activating RXRg transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra w-w stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRa-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor a （TNFα）-induced AKT activation and stimulates TNFa-mediated apoptosis in cancer cells in an RXRa/tRXRo dependent manner.The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRa to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRa ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFa-mediated cancer cell apoptosis by targeting RXRα/tRXRα.
